An important unsolved question in cell death is to understand why different cells within a population vary in their responses. Which cells will live or die and what determines exactly how they die after exposure to a death stimulus? These questions underlie fundamental cell fate decisions and also have important practical ramifications, for example, during cancer therapy when non-heritable, heterogeneous responses to anti-cancer drugs underlie the eventual acquisition of resistance to therapy. Heterogeneity in cell responses can be driven by stable genetic differences between cells, which are easy to understand. However, such differences also occur even in genetically homogeneous cell populations. What underlies these differences? More important, can we manipulate these effects? Previous work supported by this grant discovered that even in a homogeneous population of cells under unstressed conditions, there is extensive variation in the amount of autophagic flux, which in turn predicts the outcome to future treatment with a death stimulus. And, in the last funding period, we discovered a specific mechanism by which autophagy controls the apoptosis threshold and a quite different mechanism by which the autophagy machinery can control necroptosis. Building on these previous studies, we hypothesize: autophagy controls apoptotic and necroptotic thresholds by regulating Mitochondrial Outer Membrane Permeabilization (MOMP). And, this explains cell death variation between cells in a population. We will test this hypothesis by completing the following aims using a variety of new approaches including the first method that allows optogenetic regulation of autophagy. Specific Aim 1. Test if autophagy variation before and after a death stimulus controls heterogeneity in apoptosis responses in a cell population. Specific Aim 2. Determine how autophagy regulates necroptosis. By completing these aims, we will gain new insights into the interplay between two major forms of programmed cell death (apoptosis and necroptosis) and uncover how autophagy regulates these processes.